The T-cell factor (TCF) family plays key roles in the control of gene expression in response to secreted Wnt proteins. Wnt pathways operate to modulate cell proliferation, migratory behavior, cell fate determination and cell death. This proposal aims to study the regulation of TCF3, a transcriptional repressor which has been implicated in Wnt signaling during cell differentiation and tumorigenesis. Our preliminary experiments in Xenopus ectoderm and mammalian cells identified a specific post-translational change in TCF3 protein mobility that correlates with Wnt target gene activation. This effect has been demonstrated to involve homeodomain-interacting protein kinase (HIPK), leading to a hypothesis that Wnt proteins act via HIPK2 and TCF3 to regulate their transcriptional targets. This hypothesis will be tested in mammalian cells and Xenopus eggs, in which biochemical and cell biological approaches can be combined with rapid functional analysis of underlying mechanisms. To investigate roles of TCF3 postranslational modification for Wnt-dependent activation of target genes, non- phosphorylatable and phosphomimetic TCF3 mutant proteins will be compared in morphological and molecular assays. Other experiments will elucidate how Wnt signals activate HIPK and trigger TCF3 post-translational modifications that are critical for target gene regulation. These studies will help characterize basic signaling networks normally leading to cell proliferation and differentiation, but which are misregulated during disease. Given the association of Wnt pathways with cancer, the knowledge of how TCF3 is regulated will serve as a basis for new drug screens and should lead to the development of crucial therapeutic strategies. PUBLIC HEALTH RELEVANCE: This application concerns the function of TCF3, a Wnt-dependent transcriptional repressor that is essential for the regulation of cell proliferation. Since Wnt pathways have been found activated in a variety of tumors, the proposed studies are highly relevant to human cancer.